WetLab work

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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
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Dude, right on there broreht. http://unmpfo.com [url=http://fvjkubo.com]fvjkubo[/url] [link=http://uwegjh.com]uwegjh[/link]
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!align="center"|[[Team:Tsinghua|HOME]]
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!align="center"|[[Team:Tsinghua/Team|Team]]
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!align="center"|[[Team:Tsinghua/Project|Project 1]]
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!align="center"|[[Team:Tsinghua/Project2|Project 2]]
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!align="center"|[[Team:Tsinghua/Parts|Parts]]
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!align="center"|[[Team:Tsinghua/Modelling|Modelling]]
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!align="center"|[[Team:Tsinghua/Notebook|Notebook]]
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!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
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|}
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== '''CdLocalizer''' ==
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1. Reconstruct pUC18 plasmid, insert GFP to the downstream of lac promoter
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1.1 Use touchdown PCR to amplify GFP and add degradation tag and terminator.
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Primers:
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Forward: 5’- GA    GAATTC  G  AGCAAGGGCGAGGAGCTGTTCACCG -3’
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Reverse: 5’- CTTG  CCCGGG  TTATCACTTGTACAGCTCGTCCATGC -3’
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(''Template: provide by Guoqiang Chen’s lab in Tsinghua University'')
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1.2 Cut pUC18 plasmid with EcoR I and KpnI. Purify the cut pUC18 from agarose gel with Kit.
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1.3 Purify the PCR product GFP from agarose gel with Kit and cut with EcoR I and Kpn I.
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1.4 Purify the cut GFP with column.
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1.5 Run a gel to compare the concentrations of the fragment and vector in order to decide their volume in the ligation mixture.
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1.6 Incubate the ligation mixture at 16 degree.
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1.7 Use 10μl ligation mixture to transform 200μl DH5αcompetent cell, spread on the LB plate(Amp), and incubate at 37 degree over night.
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1.8 Pick three clone to 5ml LB and shake at 37 degree for 12h, miniprep the reconstruct plasmid.
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1.9 We sequenced the inserted part and it was right.
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2. Synthesize the Nde I-Pm(complementary chain)-Pst I-Xba I-cad-Spe I-RBS(strong)-CI+LVA tag-Sac II-T1, T2-BamH I (the Blue part is the synthesis sequence) sequence. We use synthesis because we cannot use PCR to get the sequence from Staphyloccocus aureus Rosenbach
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3. Insert CheZ(with tag and terminator) into the reconstruct pUC18(with GFP)
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3.1 Extract the genome of E.coli DH5αstrain and use PCR amplify CheZ from it.
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Forward primer:
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5’-GTCATGCC  CA TATG  CAACCATCAATCAAACCTGC-3’
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Reverse primer:
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5’-AT    AGG CCT  AAATCCAAGACTATCCAACAAATCGTCCACCTGATC-3’
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3.2 Purify the PCR product from gel and then cut with Nde I
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3.3 Cut the pAcYc duet-1 plasmid with EcoR V and Nde I
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3.4 Run a gel to compare the concentration of the vector and insertion in order to decide the their volumes in the ligation mixture
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3.5 Ligate the vector and the cheZ, transformate DH5α and spread the LB plate(Chl).
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3.6 Pick three clones to 5ml LB(Chl) and shake at 37 for 12h, then miniprep the reconstruct the plasmid.
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3.7 Synthesize the Stu I-tag-Hind III-T0/T1T2-AatII sequence (with stick end), which contains CheZ’s tag and terminator. We firstly synthesized the sequence as two single strings, and then annealing them together as a double string DNA.
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The two single strings’ sequence is below:
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Forward
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5’-CCT  GCTGCAAACGACGAAAACTACGCTTTAGTAGCT  TAA      A AGCTT
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Stu I end  LVA tag                              stop codon  Hind III
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CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGC  GACGT-3’
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T0                                      Aat II end
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Reverse
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5’- C  GCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCG  A AGCTT  TTA
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Aat II end      T0                                Hind III      stop codon
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(complementary)
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AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGC    AGG
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LVA tag (complementary)                  Stu I end
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3.8 Cut the pAcYcduet-1-cheZ plasmid with StuI and Aat II and column purify the vector
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3.9 Run a gel to compare the concentration of the vector(pAcYcduet-1-cheZ) and insertion (Stu I-tag-Hind III-T0/T1T2-AatII) to make sure their volumes in the ligation mixture.
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3.10 Ligate the two parts    transformation    spread plate    pick 4 clones    shake and get the reconstruct plasmid
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3.11 We sequenced it and it was right.
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3.12 Then we cut the whole CheZ-tag-terminator with NdeI and AatII and ligate the fragment into the pUC18-GFP vector, which is cut by the same two enzymes.
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3.13 This sequenced this plasmid and the sequence was right.
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4. Cut the synthesized Nde I-Pm(complementary chain)-Pst I-Xba I-cad-Spe I-RBS(strong)-CI+LVA tag-Sac II-T1, T2-BamH I (the Blue part is the synthesis sequence) sequence with BamH I and Nde I, the same was done on the vector(pUC18-cheZ-tag-ter-GFP). Then ligate them together and got a new reconstruct plasmid. It was also sequenced.
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5. Put RBS-CI-tag into last vector.
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5.1 Use PCR to amplify CI from lambda DNA, adding RBS to 5’ and tag to 3’
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First round primers:
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5’-GAGGGGACAAactagt  ATGAGCACAAAAAAGAAACCATTAACACAAGAGC-3’
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5’-TCGTTTGCTGCAGGCCT  gccaaacgtctcttcaggccactgac-3’
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Second round primers:
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5’-C  A CTAGT  tctagaGAAAGAGGGGACAAactagt  ATGAGCACAAAAAAGAAACC-3’
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5’-ACT  CCGC GG TTA AGCTGCTAAAGCGTAGTTTTCGTCGTTTGCTGCAGGCCT  gccaaacgtctcttcagg
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We also did a point mutation with PCR to mutate a Hind III restriction cite.
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Forwad:
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5’-GGCTCCAAGCCTAGCTTTCCTGACGGAATG-3’
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Reverve:
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5’- cattccgtcaggaaagctaggcttggagcc-3’
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5.2 Cut the RBS-CI-tag and the reconstructed vector with Sac II and Spe I and ligate them together.
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5.3 Transformation the final plasmid to the ΔCheZ E.coli stain.
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== '''DualReceptor''' ==
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== '''TheoRiboSwitch''' ==
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
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!align="center"|[[Team:Tsinghua|HOME]]
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-
!align="center"|[[Team:Tsinghua/Team|Team]]
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!align="center"|[[Team:Tsinghua/Project|Project 1]]
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!align="center"|[[Team:Tsinghua/Project2|Project 2]]
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!align="center"|[[Team:Tsinghua/Parts|Parts]]
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!align="center"|[[Team:Tsinghua/Modelling|Modelling]]
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!align="center"|[[Team:Tsinghua/Notebook|Notebook]]
+
-
!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
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|}
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Latest revision as of 09:26, 8 April 2016

Dude, right on there broreht. http://unmpfo.com [url=http://fvjkubo.com]fvjkubo[/url] [link=http://uwegjh.com]uwegjh[/link]

Retrieved from "http://2008.igem.org/WetLab_work"